Our laboratory?s long-range goal is to understand how HIV-1 persists despite optimal antiretroviral therapy and to develop new approaches to clear the infection. Virus isolated from patient blood encodes vpr, which is packaged into virions and expressed at late stages of viral infection. Vpr is needed for optimal infection of macrophages, tonsillar tissue and SIV model systems, however its actions in T cells are largely detrimental to infection and a requirement for Vpr for HIV infection is not observed in pure T cell systems. There is currently a critical gap in our understanding of why Vpr is conserved in the virus despite its unique role in macrophages, which are not known to be crucial for HIV transmission or persistence. Our preliminary data strongly indicate that Vpr is responsible for downmodulation of a macrophage-specific protein that interferes with Env expression. Thus, the objectives of this application are (1) to determine the extent to which Vpr-mediated downmodulation of the macrophage protein alleviates restriction of Env and spread of HIV in macrophages, (2) to determine the domain of Env required for restriction in macrophages and (3) to identify viral populations from in vivo samples that manifest these phenotypes. Our central hypothesis is that a macrophage protein binds residues on Env and targets it to degradative and/or antigen presentation compartments to restrict spread and enhance antigen presentation in infected and bystander myeloid cells; Vpr counteracts this pathway by reducing levels of the restricting protein through direct infection and fusion of Vpr-containing virions with bystander myeloid cells. The rationale for the proposed work is that a greater understanding of how HIV overcomes cellular antiviral mechanisms to establish a persistent infection in macrophages will facilitate efforts to clear infection in the CNS and other reservoirs. Thus, we plan to test our central hypothesis and accomplish the objective of this application by pursuing the following specific aims: 1. Determine the extent to which the macrophage protein we have identified accounts for restriction of Env in HIV infected macrophages lacking Vpr. 2. Define the domain of Env proteins needed for susceptibility to restriction by macrophages. 3. Identify viral populations from in vivo samples that target the restriction factor. At the completion of these studies we expect to determine the extent to which; (1) macrophages restrict viral infection by targeting a specific domain on Env, (2) sensitivity to this restriction is an important characteristic of viruses that form reservoirs in people, (3) Vpr functions to counteract this restriction by downmodulating the macrophage protein. An understanding of the macrophage restriction factor and identification of Env domains responsible for sensitivity to the restriction may provide new approaches to limit spread of HIV and enhance the immune response by promoting antigen presentation. Targeting the mechanisms by which the virus disrupts the immune system may allow the development of safer, more specific treatment approaches that will promote the clearance of HIV-1 infected cells.